Acrylonitrile (AN) is a potent toxicant and a known rodent carcinogen. AN may undergo direct conjugation with glutathione or oxidation to form cyanoethylene oxide (CEO). CEO is metabolized to yield cyanide via several proposed pathways including epoxide hydration. Further, male mice were reported to be more sensitive to the acute toxicity of AN than females. The objectives of this work include the study of the importance of CYP2E1 and EH in the metabolism of AN to cyanide and the role of these enzymes in the gender differences in AN-induced acute toxicity. Male and female wild-type (WT) and CYP2E1-null mice received AN at 0, 2.5, 10, 20, or 40 mg/kg by gavage. Blood and tissue cyanide concentrations were measured at 1 or 3 hr after dosing. Present results showed that cyanide levels in blood and tissues of AN-treated WT mice were significantly greater than controls and increased in a dose-dependent manner. In contrast, cyanide levels in the blood and tissues of AN-treated CYP2E1-null mice were not statistically different from those of vehicle-treated mice. Significantly higher levels of AN-derived cyanide were detected in the blood and tissues of WT male mice than in females, which may explain the higher sensitivity of male mice to AN-induced toxicity. Western blots of microsomal and cytosolic liver protein from WT male and female mice showed that while there was negligible differences in CYP2E1 expression, higher levels of EH were detected in male mice. Differences in the expression of EH may contribute to elevated metabolism of CEO to cyanide and to the greater sensitivity of male mice to AN-induced toxicity. In summary, these data demonstrated that CYP2E1 is the only enzyme responsible for the oxidative metabolism of AN, and suggest that metabolism of CEO via EH may play a major role in the formation of cyanide in mice. Further, gender differences in the expression of EH may explain the higher cyanide levels and the more severe toxicity observed in male vs. female mice.